Department of Biotechnology, Beijing Polytechnic, No. 9, Liang Shuihe First Street, Yi Zhuang Economic & Technological Development Zone, Beijing, 100176, China.
State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, No. 15 East Road of North Third Ring, Chao Yang District, Beijing, 100029, China.
Biotechnol Lett. 2021 Sep;43(9):1821-1830. doi: 10.1007/s10529-021-03155-5. Epub 2021 Jun 29.
There are several studies on the use of RNA interference (RNAi) for gene function analysis in fungi. However, most studies on filamentous fungi are based on in vitro-transcribed or -synthesized small interfering RNA (siRNA), and only a few have reported the use of vector-based RNAi. Here we want to develop and evaluate a new vector-based RNAi method using the mouse U6 promoter to drive short hairpin RNA (shRNA) expression in the filamentous fungi.
Molecular techniques were employed to develop and evaluate a new vector-based RNAi method using the mouse U6 promoter to drive short hairpin RNA (shRNA) expression in the filamentous fungus Blakeslea trispora.
We characterized the mouse U6 promoter and utilized it for the expression of shRNA in B. trispora. Using real-time polymerase chain reaction and western blotting analyses, we confirmed the decrease in the mRNA and protein expression of carRA, respectively, in cells transformed with the mouse U6 promoter-driven shRNA expression vector. This indicated that the shRNA was transcribed from the mouse U6 promoter and correctly processed into siRNA and that the mouse U6 promoter exhibited transcription ability in the filamentous fungi.
The results suggest that the mouse U6 promoter that drives the expression of shRNA vectors may serve as a novel tool for RNAi induction in filamentous fungi.
有几项关于 RNA 干扰 (RNAi) 在真菌基因功能分析中的应用的研究。然而，大多数丝状真菌的研究都是基于体外转录或合成的小干扰 RNA (siRNA)，只有少数报道了基于载体的 RNAi 的应用。在这里，我们希望开发和评估一种新的基于载体的 RNAi 方法，使用小鼠 U6 启动子驱动丝状真菌中的短发夹 RNA (shRNA) 表达。
采用分子技术开发和评估了一种新的基于载体的 RNAi 方法，使用小鼠 U6 启动子驱动丝状真菌 Blakeslea trispora 中的短发夹 RNA (shRNA) 表达。
我们对小鼠 U6 启动子进行了表征，并利用其在 B. trispora 中表达 shRNA。通过实时聚合酶链反应和 Western blot 分析，我们分别证实了细胞中 carRA 的 mRNA 和蛋白表达降低，这些细胞被转化为小鼠 U6 启动子驱动的 shRNA 表达载体。这表明 shRNA 是由小鼠 U6 启动子转录的，并正确地加工成 siRNA，而且小鼠 U6 启动子在丝状真菌中有转录能力。
结果表明，驱动 shRNA 载体表达的小鼠 U6 启动子可能成为丝状真菌中 RNAi 诱导的一种新工具。
